One of the first steps of DNA replication is the loading of the replication proteins onto the DNA that is to be replicated. The T4 gene 59 protein binds DNA and plays a key role in this process, by greatly stimulating the loading of T4 gene 41 helicase at the site of replication, leading to the formation of the complete protein complex necessary for efficient replication of the T4 genome. Similar events are thought to happen during the initiation of DNA replication in all organisms, so the insight gained through studying the biochemistry of gene 59 loading protein may help to illuminate general mechanisms involved in DNA replication. Efficient T4 DNA replication also requires the functions of another DNA binding protein, the 32 single-stranded binding protein (SSB). At least two models have emerged to describe the interactions between these two proteins during the assembly of processive replisomes at the T4 origins. In one model 32 SSB and 59 protein form a complex that recruits the replicative 41 helicase to the origins. In the second model, 32 SSB must first be wiped off the origins by an accessory helicase or displaced by 59 protein before 41 can be loaded and the replisome assembled. We directly tested these models in vitro using a panel of 59 mutants and 32 truncations and determined that 32 SSB inhibits 59 mediated helicase loading on forked DNA substrates designed to mimic origins. This inhibition was observed with both 32-B, a 32 SSB truncation that does not bind DNA, and 32-A, which retains DNA binding activity. Moreover, a 59 mutant deficient in 32 interactions is not affected by 32-B but is by 32-A. Thus, it appears that 32 SSB can inhibit 59 mediated helicase loading through two separate mechanisms, competition for fork DNA and direct interaction with 59 in solution. The net effect is that 32 SSB disrupts formation complexes between 59 protein and 41 helicase on forked DNA, implying that there is no loading competent, ternary complex formed between the three proteins. Yet, complexes between 32 SSB and 59 protein do have a functional significance during T4 DNA replication. 32 can recruit 59 protein to single-stranded DNA, an activity that requires direct interaction with 59 protein. If this interaction is disrupted by mutation of 59, T4 DNA replication is disrupted, and abnormally large lagging strand fragments are synthesized. Our results suggest that 59 has at least two functional roles during T4 DNA replication, facilitating 41 helicase loading at the origins and organizing lagging strand synthesis as the replisome moves along the chromosome.[unreadable] [unreadable] Although T4 59 protein is thought to actively load the replicative 41 helicase during replisome assembly at the viral origins of replication, some T4 origins are active during infection in the absence of 59 protein, This indicates that 59 protein is not absolutely necessary for helicase loading. To determine exactly what biochemical activities are necessary for normal T4 replication we investigated the effects of several defined 59 mutants on in vitro replication and viral DNA synthesis during infection. As expected, a 59 mutant deficient in DNA binding was incapable of stimulating helicase loading and T4 DNA replication, both in vitro and during infection. Yet, the 59 mutant deficient in helicase interaction, which was unable to efficiently load 41 helicase or stimulate 41 dependent DNA replication in vitro, had little obvious effect on viral replication during infection. Both the total amount of DNA synthesized over the course of infection and the pattern of replication across the T4 chromosome were very similar to normal infections. The replication observed with this 59 mutant was almost entirely dependent on T4 dda helicase, implying that this accessory helicase is involved in 41 helicase loading. Another 59 mutant deficient in interactions with 32 single-stranded DNA binding protein (SSB) had a different effect on T4 replication. This mutant caused a lag in total DNA synthesis, both in vitro and during infection, and the pattern of replication was devoid of the typical peaks of DNA synthesis near the origins. The reduction in origin synthesis is apparently caused by a defect in 59 gatekeeping activity, holding T4 DNA polymerase in place until 41 helicase is loaded onto the origins. Whereas the 59 mutant deficient in helicase interactions has normal gatekeeping activity on 32 SSB coated substrates, the 59 mutant deficient in 32 SSB interactions does not. Hence, it appears that the primary function of 59 protein during infection is to target replisome assembly to the T4 origins.